Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical and allied health fields. A particularly important area is in mammalian including human genetics and the molecular mechanisms behind some genetic abnormalities. Progress in research in this area has been hampered by the lack of a cloned nucleic acid molecule encompassing a human centromere. The identification and cloning of a human centromere will promote the development of techniques for introducing genes into eukaryotic cells and in particular mammalian including human cells and will be an important asset to gene therapy and the development of a range of genetic diagnostic tests.
The centromere is an essential structure for sister chromatid cohesion and proper chromosomal segregation during mitotic and meiotic cell divisions. The centromere of the budding yeast Saccharomyces cerevisiae has been extensively studied and shown to be contained within a relatively short DNA segment of 125 bp that is organized into an 8-bp (CDEI) and 26-bp (CDEIII) domain, separated by a 78- to 87-bp, highly AT-rich, middle (CDEII) domain (Clarke and Carbon, 1985). The centromere of the fission yeast Schizosaccharomyces pombe is considerably larger, ranging from 40 to 100 kb, and consists of a central core DNA element of 4 to 7 kb flanked on both sides by inverted repeat units (Steiner et al., 1993). Recently, the functional DNA components of a higher eukaryotic centromere have been characterized in a minichromosome from Drosophila melanogaster and shown to consist of a 220-kb essential core DNA flanked by 200 kb of highly repeated sequences on one side (Murphy and Karpen, 1995).
The mammalian centromere, like the centromeres of all higher eukaryotes studied to date, contains a great abundance of highly repetitive, heterochromatic DNA. For example, a typical human centromere contains 2 to 4 Mb of the 171-bp α-satellite repeat (Wevrick and Willard 1989, 1991; Trowell et al., 1993), plus a smaller and more variable quantity of a 5-bp satellite III DNA (Grady et al., 1992; Trowell et al., 1993). The role of these satellite sequences is presently unclear. Transfection of a cloned 17-kb uninterrupted α-satellite array into cultured simian cells (Haaf et al., 1992) or a 120-kb α-satellite-containing YAC into human and hamster cells (Larin et al., 1994) appear to confer centromere function at the sites of integration. Other workers have analyzed rearranged Y chromosomes (Tyler-Smith et al., 1993), or dissected the centromere of the human Y chromosome with cloned telomeric DNA (Brown et al., 1994) and suggested that 150 to 200 kb of α-satellite DNA plus ˜300 kb of adjacent sequences are associated with human centromere function. In addition, a human X-derived minichromosome that retained 2.5 Mb of α-satellite array has been produced by telomere-associated chromosome fragmentation (Farr et al., 1995). In all these studies, it is not known whether non-α-satellite DNA sequences are embedded within the centromeric site and operate independently of, or in concert with, the α-satellite DNA.
In mammals, four constitutive centromere-binding proteins, CENP-A, CENP-B, CENP-C, and CENP-D, have been characterized to varying extents and implicated to have possible direct roles in centromere function. CENP-A, a protein localized to the outer kinetochore domain, is a centromere-specific core histone that shows sequence homology to the histone H3 protein and may serve to differentiate the centromere from the rest of the chromosome at the most fundamental level of chromatin structure—the nucleosome (Sullivan et al., 1994). CENP-B, a protein which associates with the centromeric heterochromatin through its binding to the CENP-B box motif found in primate α-satellite and mouse minor satellite DNA, probably has a role in packaging centromeric heterochromatic DNA—a role which, however, may not be indispensable since the protein is undetectable on the Y chromosome (Pluta et al., 1990) and is found on the inactive centromeres of dicentric chromosomes (Earnshaw et al., 1989). CENP-C has been shown to be located at the inner kinetochore plate and is postulated to have an essential although yet undetermined centromere function, as seen, for example, from inhibition of mitotic progression following microinjection of anti-CENP-C antibodies into cells (Bernat et al., 1990; Tomkiel et al., 1994) and from its association with the active but not the inactive centromeres of dicentric chromosomes (Earnshaw et al., 1989; Page et al., 1995; Sullivan and Schwartz, 1995). Finally, CENP-D (or RCC1) is a guanine exchange factor that appears to have a general cellular role that is neither specific nor clear for the centromere (Kingwell and Rattner 1987; Bischoff et al., 1990; Dasso, 1993). More recently, a new role for the mammalian centromere as a “marshalling station” for a host of “passenger proteins” (such as INCENPs, MCAK, CENP-E, CENP-F, 3F3/2 antigens, and cytoplasmic dynein), has been recognized (reviewed by Earnshaw and Mackay, 1994, and Pluta et al., 1995). These passenger proteins, whose appearance at the centromere is transient and tightly regulated by the cell cycle, provide vital functions that include motor movement of chromosomes, modulation of spindle dynamics, nuclear organization, intercellular bridge structure and function, sister chromatid cohesion and release, and cytokinesis. At present, except for CENP-B, none of the constitutive or passenger proteins have been demonstrated to bind mammalian centromere DNA directly.
In work leading up to the present invention, the inventors identified in a patient (hereinafter referred to as “BE”) an unusual human marker chromosome, mardel 10, which is 100% stable in mitotic division both in patient BE and in established fibroblast and transformed lymphoblast cultures. In accordance with the present invention, a region of the mardel (10) chromosome has been cloned together with the corresponding region from a normal human subject. The nucleic acid molecules cloned contain no substantial α-satellite repeats yet are mitotically stable. The nucleic acid molecules encompass therefore, a new form of centromere referred to herein as a “neocentromere”. The identification and cloning of a eukaryotic neocentromere without substantial α-satellite DNA repeat sequences now provides the means of generating a range of eukaryotic artificial chromosomes such as mammalian including human artificial chromosomes with uses in genetic therapy, transgenic plant and animal production and recombinant protein production. A range of diagnostic reagents is now also obtainable using the cloned neocentromere.